Well unit for detecting cell chemotaxis and separating chemotactic cells

ABSTRACT

The present invention aims at providing a well unit to be used in fabricating an apparatus whereby movements of cells based on their own actions can be accurately and easily detected, in detecting the chemotaxis of cells due to a chemotactic factor or the inhibition of the chemotaxis of cells by an inhibitor.  
     Accordingly, the present invention provides a well unit to be used in an apparatus for detecting chemotaxis of cells and separating cells characterized in that a plural number of wells, in which a liquid sample can be held in a resting state, are connected to each other via a channel, the channel is provided with a bank, and, in the upper part of the bank, barriers constituting one or more grooves having a width and/or a depth fit for the diameter or deformability of cells are provided or a plane is provided so as to give a gap having a depth fit for the diameter or deformability of cells between the plane and the glass substrate.

TECHNICAL FIELD

[0001] This invention relates to a well unit to be used in an apparatusfor judging whether or not cells can migrate in a definite direction bytheir own actions, observing the state of cells migrating in a definitedirection by their own actions, or counting cells having migrated in adefinite direction by their own actions (i.e., an apparatus fordetecting chemotaxis of cells) and an apparatus for separating cellsbased on the selective migration of cells in a definite direction bytheir own actions.

BACKGROUND ART

[0002] It has been a practice to use a Boyden chamber as an apparatusfor detecting chemotaxis of cells in vitro. This apparatus has astructure partitioned into an upper chamber and a lower chamber by afilter having pores (diameter: 3 to 8 μm) through which cells can pass.A cell suspension is put into the upper chamber while a specimensolution containing a chemotactic factor is put into the lower chamber.Then cells migrating toward the chemotactic factor through the filter orcells appearing on the back face of the filter are counted. In thisapparatus which is most commonly employed today, it is necessary to use¼ to {fraction (1/20)} ml of a cell suspension having a concentration of1×10⁶ cells/ml, i.e., corresponding to at least 5×10⁴ cells. Althoughthere scarcely arise any problems in case of analyzing cells which canbe obtained in large quantities, it is highly laborious to obtain anecessary amount of cells occurring at a very limited level, forexample, eosinophils contained in an amount of about 1 to 5% inperipheral leukocytes, basophils contained in an amount of 1% or lesstherein, or monocytes contained in an amount of about 1 to 2% therein.In case of using a small animal such as a mouse, blood can be collectedin a highly limited amount, i.e., about 1.0 ml per animal at thelargest. Moreover, cancer cells and some of cells existing in tissuescan be hardly obtained in a large amount and it is therefore desired toexamine the characteristics of these cells in microquantities.Furthermore, the Boyden chamber suffers from an additional problem thatcells in the course of migration cannot be observed or counted thereby.

[0003] There have been marketed slide glass plates for qualitativeanalyses by which chemotaxis of cells can be observed at a level ofseveral individuals. In such a slide glass plate, two grooves (wells) of4 mminwidth, 25 mm in length and 1 mm in depth are formed in both sidesof a bridge (channel) of 1 mm in width on a glass slide (25×75 mm, 2 mmin thickness) for microscopes. Namely, two wells are connected to eachother via the channel. A cell suspension is put into one well and aspecimen solution containing a chemotactic factor is put into the otherwell. After covering with a glass plate, cells migrating from one wellto the other well across the channel are observed. In this case,however, it is not assumed that the bridge forms a gap fitting for thediameter or derformability of the cells. Also, no groove through whichthe cells pass is formed in the channel. In addition, each well has acapacity of 100 μl. That is to say, it is needed to use at least{fraction (1/10)} ml of a cell suspension per well. Also, there has beenmarketed another chemotaxis chamber having a similar structure in whichtwo grooves (wells) are concentrically formed on a slide glass plate anda bridge (channel) is provided between these grooves (Dun ChemotaxisChamber® manufactured by Waber Scientific). In this case, a cellsuspension is put into the inner well while a specimen is put into theouter well. After covering with a glass plate, cells passing through thechannel are microscopically observed. The channels are located lower by20 μm than the cover glass and cells pass through the gap between them.The distance between the channel plane and the cover glass is setregardless of the diameter or deformability of cells and the channelshave no grooves through which cells pass.

[0004] To measure blood rheology, Kikuchi et al. have proposed anapparatus having channels provided with a plural number of microgroovesformed on the surface of a single-crystal silicon substrate by usingsemiconductor fabrication techniques (Kikuchi, et. al., SPIE Vol.2978,165-171 (1997); Kikuchi, et. al., Microvascular Research, Vol.44,226-240 (1992); Kikuchi, et. al., Seibutsu Butsuri (Biophysics),Vol.214, 254-258 (1997)). In this apparatus, it is intended to make ablood cell suspension flow due to a difference in pressure between bothsides of the channel thereby observing and studying the blood flow.Although behaviors can be observed thereby at the cellular level, nostructure for observing or measuring migration of blood cells by theirown actions is employed in this idea.

[0005] Japanese Patent No. 2532707 has disclosed a blood circuit whereinlarge grooves each having an entrance port at one end and an exist portat the other end are formed in parallel and barriers partitioning thesegrooves are provided with microgrooves, by which the large grooves areconnected to each other, orthogonally to the lines connecting theentrance ports to the exist ports. In this circuit, a blood sample isflown in one of the large grooves while a specimen containing achemotactic factor is flown in the other groove. Then a portion of theblood sample is introduced into the microgrooves (channels) and cellspassing through the microgrooves (channels) are detected to therebyexamine the movements and functions of the cells or observe and measurethe mobility thereof. Since flows in which the blood sample and thechemotactic factor-containing specimen are circulated are formed by thelarge grooves, this circuit has no well in which the blood sample or thechemotactic factor-containing specimen is contained in a resting state.In addition, the blood sample and the chemotactic factor-containingspecimen are required each in a considerably large amount. Accordingly,this apparatus is unsuitable for studying movements of cells by theirown actions with the use of microsamples.

[0006] There has been also known a blood filter wherein cells in bloodare passed thorough microgrooves and thus the state of the blood cellsduring passage is observed (Japanese Patent No. 2685544). This filterconsists of a first substrate made of a silicone substrate havingmicrogrooves on the surface and a second substrate having a planejointed to the surface of the first substrate. Blood cells pass througha space formed by the grooves of the first substrate at the interface ofthese substrates. To make the flow of blood cells in the microgrooves,it is needed to apply an external force by pressurizing, sucking, etc.Accordingly, the flow of the cells by their own actions cannot beobserved by this apparatus. Namely, this apparatus has no well in whicha blood sample or a specimen solution is contained in a resting state.

[0007] To fractionate cells depending on functional properties such ascell membrane hardness or cell deformability, there have been also knownapparatuses by which cells to be fractionated are passed thoroughchannels having a large number of microgrooves to thereby divide thecells into passable ones and non-passable ones. For example, JapanesePatent No. 2685119 has proposed an apparatus wherein channels havingdifferent groove widths are formed in two stages for the multistagefractionation of cells. However, a solution containing cells is migratedunder elevated pressure in this apparatus and thus migration of cells bytheir own actions cannot be understood thereby.

[0008] Moreover, there has been known a laminated microchannel arrayapparatus wherein substrates having channels provided with microgroovesare piled up each other so as to enable the filtration and fractionationof a large amount of a cell suspension (Japanese Patent Laid-Open No.165062/1999). However, a solution containing cells is migrated underelevated pressure in this apparatus too and thus migration of cells bytheir own actions cannot be understood thereby.

DISCLOSURE OF THE INVENTION

[0009] The present invention aims at providing a well unit to be used inan apparatus whereby movements of cells based on their own actions canbe accurately and easily detected, in case of detecting the chemotaxisof cells due to a chemotactic factor or the inhibition of the chemotaxisof cells by an inhibitor. The term “movements based on their ownactions” as used herein means that cells migrate by their own actionswithout being affected by, for example, pressure. This is an importantfactor in examining and confirming the effect of a chemotactic factor ata high reliability. To accurately detect such movements of cells bytheir own actions, it is highly required that the cells are broughttogether in the vicinity of channels and aligned in the flow directionof the cells before the initiation of the migration. However, there hasbeen known no well unit structure by which the above-described situationcan be established in microwells.

[0010] The present invention further aims at providing a well unit to beused in an apparatus for detecting the chemotaxis of cells by using cellsamples in microquantities. In addition, the present invention aims atproviding a well unit to be used in an apparatus for efficientlysearching for a chemotactic substance and an inhibitor thereof with theuse of a large number of specimens at once. The present inventionfurthermore aims at providing a well unit to be used in an apparatus forseparating and collecting specific cells from a liquid mixturecontaining cells of plural types.

[0011] Accordingly, the present invention relates to a well unit to beused in an apparatus for detecting chemotaxis of cells and separatingcells characterized in that a plural number of wells, in which a liquidsample can be held in a resting state, are connected to each other via achannel, the channel is provided with a bank, the wells are formed so asto tightly bond to a glass substrate, and, in the upper part of thebank, barriers constituting one or more grooves having a width and/or adepth fit for the diameter or deformability of cells are provided, or aplane is provided so as to give a gap having a depth fit for thediameter or deformability of cells between the plane and the glasssubstrate; and to the well unit as described above characterized in thata plural number of wells, in which a liquid sample can be held in aresting state, are connected to each other via a channel, the channel isprovided with a bank and, in the upper part of the bank, barriersconstituting one or more grooves having a width and/or a depth fit forthe diameter or deformability of cells are provided. By providing thebank or by providing these barriers constituting the grooves, it ispossible to easily bring together the cells held in the wells in thevicinity of the channel and align them in the flow direction of thecells before the initiation of migration. This effect can be furtherenhanced by employing a structure wherein a barrier for restricting themigration of the cells in the step of aligning the cells along the startline is formed orthogonally to the direction toward the opposite well,or a barrier for restricting the migration of the cells in the step ofaligning the cells along the start line is formed in parallel to thearray of the barriers.

[0012] In this well unit, a plural number of wells may be connected inseries to each other each via a channel. Alternatively, a plural numberof wells may be connected to a single well each via a channel.Alternatively, among a plural number of wells connected to a single welleach via a channel, at least two wells are connected to another commonwell each via a channel.

[0013] A plural number of wells as described above are wells holding acell suspension and wells holding a solution containing a chemotacticfactor. Alternatively, these wells are wells holding a cell suspension,wells holding a solution containing a chemotactic factor, and wellsholding a solution containing a chemotactic factor inhibitor.

[0014] In the well unit according to the present invention, it is alsopossible to provide a wall orthogonal to the channel in one or both ofwells connected to each other via the channel to thereby restrict theamount of the liquid in the vicinity of the channel. In this well unit,moreover, a terrace may be formed to one or both of the walls formedorthogonally to the channel.

[0015] It is also possible to give a screen-positioning mark fordetecting cells on any point in the upper part of a bank in the wellunit according to the present invention. Furthermore, a multistage bankmay be formed in the channel.

[0016] In the well unit according to the present invention, the groovesformed in the channel may be connected to each other via one or moregrooves orthogonal to the direction toward the opposite well.Alternatively, it is also possible that the width of a plural number ofgrooves in the direction toward the opposite well in the channel ischanged stepwise each time the grooves intersect one or more groovesorthogonal thereto, or a plural number of grooves in the directiontoward the opposite well in the channel are formed by mutually shiftingthe positions thereof each time the grooves intersect one or moregrooves orthogonal thereto.

[0017] In the channel in the well unit according to the presentinvention, it is also possible that terraces are formed in the front andthe rear of an array of barriers constituting one or more grooves havinga width and/or a depth fit for the diameter or deformability of cells inthe channel and the terrace in the cell flow direction is longer thanthe other terrace.

[0018] In the channel in the well unit according to the presentinvention, a terrace may be formed at the center in the channel, arraysof walls constituting one or more grooves having a width and/or a depthfit for the diameter or deformability of cells may be formed at twopositions in both sides of the terrace, and, if desired, terraces may befurther formed outside the barrier arrays.

[0019] It is also possible to refer each of the well units as describedabove to as a single unit and integrate a plural number of units of oneor more types to thereby give a well unit for detecting chemotaxis ofcells and separating cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a sectional view which shows an example of the mode ofusing the well unit according to the present invention.

[0021]FIG. 2 is a top plan view which shows an example of the well unitaccording to the present invention.

[0022]FIG. 3 is a sectional view which shows an example of the mode ofusing the well unit according to the present invention.

[0023]FIG. 4 is a top plan view which shows an example of the well unitaccording to the present invention.

[0024]FIG. 5 shows another example of the mode of using the well unitaccording to the present invention, wherein (1) is a sectional view ofan apparatus; and (2) is a top plan view which shows an example of thewell unit according to the present invention.

[0025]FIG. 6 shows another example of the mode of using the well unitaccording to the present invention, wherein (1) is a sectional view ofan apparatus; and (2) is a top plan view which shows an example of thewell unit according to the present invention.

[0026]FIG. 7 shows an example of the well unit according to the presentinvention wherein three wells are connected in series to each other eachvia a channel.

[0027]FIG. 8 shows an example of the well unit according to the presentinvention wherein three wells having penetrating holes are connected inseries to each other each via a channel.

[0028]FIG. 9 shows an example of the well unit according to the presentinvention wherein a plural number of wells are connected to a singlewell each via a channel.

[0029]FIG. 10 shows an example of the well unit according to the presentinvention wherein a plural number of wells are connected to a singlewell each via a channel and the wells have penetrating holes.

[0030]FIG. 11 shows an example of the well unit according to the presentinvention wherein a plural number of wells having penetrating holes areconnected to a single well each via a channel.

[0031]FIG. 12 shows an example of the well unit according to the presentinvention wherein a plural number of wells are connected to each othereach via a channel around a single well as located at the center,thereby forming a circular structure as a whole. In this figure, thewells have penetrating holes.

[0032]FIG. 13 shows an example of the well unit according to the presentinvention wherein a plural number of wells are connected to each othereach via a channel around a single well located at the center, therebyforming a circular structure as a whole.

[0033]FIG. 14 shows an example wherein a plural number of wells areconnected to each other each via a channel around a single well locatedat the center and, among these wells, each couple of wells are connectedto another common well each via a channel. In this figure, the wellshave penetrating holes.

[0034]FIG. 15 shows an example of the well unit having walls providedorthogonally to the channel.

[0035]FIG. 16 shows other examples of the well unit having wallsprovided orthogonally to the channel.

[0036]FIG. 17 shows an example of the channel structure.

[0037]FIG. 18 shows another example of the channel structure.

[0038]FIG. 19 shows an example of the arrangement of barriers in thechannel wherein the arrow shows the direction toward the opposite well.

[0039]FIG. 2 is a sectional view of the barrier arrangement shown byFIG. 19.

[0040]FIG. 21 shows an example wherein grooves in the direction towardthe opposite well across the channel are connected to each other viaanother groove formed orthogonally thereto. In this figure, each arrowshows the direction toward the opposite well.

[0041]FIG. 22 shows an example wherein grooves in the direction towardthe opposite well across the channel are connected to each other viagrooves formed orthogonally thereto. In this figure, the arrow shows thedirection toward the opposite well.

[0042]FIG. 23 shows an example wherein grooves in the direction towardthe opposite well across the channel are connected to each other via twogrooves formed orthogonally thereto and the width of the grooves in thedirection toward the opposite well is changed stepwise each time thegrooves intersect the grooves orthogonal thereto. In this figure, eacharrow shows the direction toward the opposite well.

[0043]FIG. 24 shows an example of the modification of the well unit ofFIG. 8 in which the barriers have the same size but are changed innumber. In this figure, the arrow shows the direction toward theopposite well.

[0044]FIG. 25 shows an example wherein grooves in the direction towardthe opposite well across the channel are connected to each other viathree grooves formed orthogonally thereto and the grooves in thedirection toward the opposite well are formed by mutually shifting thepositions thereof each time the grooves intersect the grooves orthogonalthereto. In this figure, the grooves shift by ½ pitch toward theorthogonal direction. Each arrow shows the direction toward the oppositewell.

[0045]FIG. 26 shows an example wherein barriers are jointed in thedirection toward the opposite well. In this figure, each arrow shows thedirection toward the opposite well.

[0046]FIG. 27 shows an example wherein terraces are formed on both sideof the array of barriers and one of the terrace is longer than theother. In this figure, the arrow shows the direction toward the oppositewell.

[0047]FIG. 28 shows an example wherein a terrace is formed at the centerof a bank and arrays of barriers are formed at two positions in bothsides of the terrace.

[0048]FIG. 29 shows an example of the well unit having walls formedorthogonally to channels wherein a terrace is formed to the walls in thechannel.

[0049]FIG. 30 shows another example of the well unit having walls formedorthogonally to the channel wherein a terrace is formed to the wall inthe channel.

[0050]FIG. 31 shows an example of the well unit having a wall formed inexclusively one of wells wherein a terrace is formed to the wall in thechannel.

[0051]FIG. 32 shows an example wherein a multistage bank is formed inthe channel.

[0052]FIG. 33 shows an example of an integration of multiplicity ofunits wherein the units are all in the same type.

[0053]FIG. 34 shows an example of an integration of multiplicity ofunits wherein the units are all in the same type.

[0054]FIG. 35 shows an example of an integration of multiplicity ofunits wherein the units of FIG. 15 are integrated.

[0055]FIG. 36 shows an example of an integration of multiplicity ofunits wherein the units are integrated circularly.

[0056]FIG. 37 shows an example of an integration of multiplicity ofunits wherein the units are in different types.

[0057]FIG. 38 shows an example of a process for constructing a channeland wells.

[0058]FIG. 39 shows an example of the fabrication of an apparatus fordetecting chemotaxis of cells and separating chemotactic cells wherein(1) provides perspective views of individual parts and (2) providessectional views corresponding thereto.

[0059]FIG. 40 shows an example wherein an obstacle is formed on a bankto thereby restrict cell migration.

DESCRIPTION OF THE REFERENCE NUMERALS AND SIGNS

[0060]1: channel.

[0061]2: well. Appendixes A, B, B_(1−n) and C are provided todifferentiate the wells (the same applies hereinafter).

[0062]3: tube for injecting/collecting samples. Appendix a represents apenetrating hole corresponding to a tube 3. Appendix b represents thetop end of the tube 3.

[0063]4: tube for avoiding increase/decrease in pressure atinjecting/collecting samples. Appendix a represents a penetrating holecorresponding to a tube 4. Appendix b represents the top end of the tube4.

[0064]5: groove in the direction toward the opposite well acrosschannel.

[0065]6: barrier.

[0066]7: substrate.

[0067]8: glass substrate.

[0068]9: block having tube mounted thereto.

[0069]10: bank.

[0070]11 11 _(−1 to 4): terraces.

[0071]12: groove orthogonal to the groove 5.

[0072]13: detector.

[0073]14: wall formed orthogonally to channel.

[0074]15: space held together by the top ends of the tubes 3 and 4.

[0075]16: packing.

[0076]17: cover cap.

[0077]18: O-ring.

[0078]19: guide pin receiver hole.

[0079]20: guide pin.

[0080]21: intermediate base.

[0081]22: bottom base.

[0082]23: mark for screen positioning.

[0083]24: obstacle.

BEST MODE FOR CARRYING OUT THE INVENTION

[0084] The well unit to be used in an apparatus for detecting chemotaxisof cells and separating chemotactic cells according to the presentinvention has a structure wherein a plural number of wells are jointedand connected to each other via a channel. The term “well” as usedherein means a container in which a cell suspension or a sample solutioncontaining, for example, a chemotactic factor or a chemotactic factorinhibitor is held. The term “channel” means a part by which two wellsare connected to each other and through which cells migrate from onewell to the other well. As will be described hereinafter, the channel isprovided with a bank and, in the upper part of the bank, barriersconstituting one or more grooves having a width and/or a depth fit forthe diameter or deformability of cells are provided, or a plane isprovided so as to give a gap fit for the diameter or deformability ofcells between the plane and the glass substrate. The term“deformability” of cells means that, in case of flexible cells, thecells can easily change their shape (for example, into flat orstring-shaped cells) owing to the flexibility and thus can pass througha gap having a smaller size than the diameter of the cells being in theinherent spherical shape in a free space.

[0085] By forming the above gap, cells migrate beyond the obstacle(i.e., the gap). Namely, the migration of the cells by their own actionsis interfered so that the chemotaxis of the cells can be more accuratelyjudged. Such a gap can be obtained by forming a bank or forming barriersconstituting grooves on the bank. Thus, it becomes possible to easilyestablish the state wherein cells contained in the wells are broughttogether in the vicinity of the channel and aligned in the flowdirection of the cells before the initiation of the migration. In caseof forming grooves through which individual cells pass, it is possibleto observe individual cells and thus the cells can be classifieddepending on desired types.

[0086] The present invention relates to a manner of connecting the wellsto be used in the above-described apparatus for detecting chemotaxis ofcells and separating chemotactic cells, the well structure and thechannel structure.

[0087]FIG. 1 shows an example of an apparatus for detecting chemotaxisof cells and separating chemotactic cells having the well unit accordingto the present invention. FIG. 2 is a top plan view of the well unitemployed in the apparatus of FIG. 1. In the examples shown by thesefigures, the well unit has wells 2A and 2B in which a cell suspension, aspecimen solution, etc. is contained. In the upper face of a bank 10partitioning these wells, a barrier 6 forming a groove 5 is provided.This well unit is tightly covered with an optically transparent glasssubstrate 8 to thereby form an enclosed space. In the presentdescription, a partial structure involving the bank 10 and the barrier 6constituting the groove 5 is called a channel. When a cell suspension issupplied into one (2A) of the wells 2, the cells tend to migrate towardthe other well (2B) and thus pass through the channel in case where thewell 2B contains a specimen solution of a chemotactic factor. Themigration state of the cells can be observed by a detector 13, forexample, a microscope. In another example of using this apparatus, asuspension of a cell mixture containing various cells is put into thewell 2A and a specific chemotactic factor is introduced into the well2B. Then cells migrating from the well 2A to the well 2B are collected.Thus, cells reacting with the chemotactic factor can be selectivelyseparated.

[0088]FIG. 3 shows another example of an apparatus for detectingchemotaxis of cells and separating chemotactic cells having the wellunit according to the present invention. The well unit has a channel 1and wells 2A and 2B in which a sample such as a cell suspension or aspecimen solution is contained. A sample is supplied into the well 2A or2B through a tube 3A or 3B with the use of a micropipette, etc. Afterthe migration, cells are collected from the well 2A or 2B through thetube 3A or 3B with a micropipette, etc. too.

[0089] When a cell suspension (i.e., one of samples) is supplied intothe well 2A through the tube 3A with a micropipette or the like, therearises a phenomenon that cells pass through the channel 1 and thus enterinto the well 2B due to the injection liquid pressure, which bringsabout confusion in the judgment whether or not the migration of thecells is caused by the chemotaxis of the specimen. In case where it isintended to separate cells, moreover, the desired cells are contaminatedwith other cells and thus the object cannot be achieved. When a specimensolution is supplied into the well 2B through the tube 3B with amicropipette or the like, there also arises a phenomenon that cells passthrough the channel 1 and thus enter into the well 2A due to theinjection liquid pressure, thereby contaminating the cell suspensiontherein. Thus, the passage of the cells through the channel 1 owing tothe chemotaxis thereof is disturbed or inhibited.

[0090] To solve this problem, another tubes 4 are provided in connectionto the tubes 3 respectively. In this structure, the injection pressureapplied on the tubes 3 is relieved in the direction of the tubes 4 andthus the forced passage of the cells toward the channel 1 can beprevented. By providing the tubes 4 connected to the tubes 3 throughwhich a sample is injected, the effect of the liquid pressure in thehorizontal direction can be minimized and thus it can be accuratelyjudged whether or not the specimen solution has chemotaxis. Relief ofthe pressure difference by the tubes 4 is also effective in relievingthe reduction in pressure in the step of collecting a sample such ascells from the wells. Thus the collection of the sample can befacilitated.

[0091] The present invention provides a well unit usable in such anapparatus. FIG. 4 shows an example of the well unit according to thepresent invention which is usable in the above-described apparatus. Awell 2A has penetrating holes 3Aa and 4Aa respectively for mountingtubes 3A and 4A, while another well 2B has penetrating holes 3Ba and 4Barespectively for mounting tubes 3B and 4B.

[0092]FIG. 5(1) and (2) show other examples of an apparatus fordetecting chemotaxis of cells and separating chemotactic cells hayingthe well unit according to the present invention. In the apparatus ofFIG. 5, a space 15 held in common by the top ends 3Ab and 3Bb of tubes3A and 3B is formed so as to lessen the effect of the pressure in thestep of injecting a sample into the wells or collecting the sample fromthe wells, as shown by FIG. 5(1). By filling up the wells 2A and 2B, thetubes 3A and 3B and the space 15 with a liquid not affecting the samplesuch as cells, the whole unit is maintained under a definite pressure.In the step of injecting or collecting the sample through the tube 3A or3B, therefore, the pressure changes in the horizontal direction can berelieved.

[0093] The well unit provided by the present invention involves a wellunit usable in such an apparatus. FIG. 5(2) shows an example of the wellunit according to the present invention which is usable in theabove-described apparatus. A well 2A has a penetrating hole 3Aa formounting a tube 3A, while another well 2B has a penetrating hole 3Ba formounting a tube 3B.

[0094] In an example of the application of the apparatus shown by FIG.5, tubes 3A and 3B and connecting tubes 4A and 4B may be formed in wells2A and 2B and a space for holding a liquid may be formed on the top endsof these tubes, as shown by FIG. 6(1). As FIG. 6(2) shows, each well hastwo penetrating holes in the well unit according to the presentinvention employed in this case. FIG. 6 shows an example of using a wellunit in which no barrier is formed on a bank 10 in a channel 1.

[0095] In the well unit according to the present invention, a pluralnumber of wells can be jointed and connected to each other in variousmanners depending on the purpose. Well units wherein a plural number ofwells 2 are jointed and connected to each other each via a channel 1 invarious manners are involved in the scope of the present invention (seeFIGS. 7 to 14).

[0096] In the present invention, moreover, it is also possible to designthe well structure concerning the channel so that chemotaxis can beexamined with the use of as a small amount of cells as possible (seeFIGS. 15 and 16).

[0097] In the channel 1, it is preferable to form barriers constitutingone or more (for example, about 20 to about 100) grooves having a widthand/or a depth fit for the diameter or deformability of cells. Byproviding these grooves, it becomes possible to control the diffusion ofcells or specimens and thus the movements of cells can be moreaccurately observed at the level of individual cells. Formation of thesegrooves also facilitates the position-adjustment of cells in the wells.That is to say, it becomes possible to easily achieve the state whereinthe cells are brought together in the vicinity of the channel andaligned in the flow direction of the cells before the initiation of themigration. In the well unit according to the present invention, variousbarrier structures may be employed depending on the purpose (see FIGS.19 to 25).

[0098] A terrace may be formed in the channel 1. By altering the terracestructure, cells having passed through the channel or under passage canbe easily observed. It is also possible to control the position of cellsto the channel in a well. The present invention relates to the wellunits having these terrace structures (see FIGS. 26 to 31).

[0099] The well unit according to the present invention involves in itsscope an integration of a plural number of units, referring wellsjointed via a channel as a single unit (see FIGS. 33 to 37). Integrationof the well units makes it possible to fabricate an apparatus fordetecting chemotaxis of cells and separating cells whereby plural typesof cells or specimens can be treated at once.

[0100] In an apparatus for detecting chemotaxis of cells and separatingcells having the well unit according to the present invention, themigration of cells can be observed and the cells under passage through achannel or having passed therethrough can be counted by providing thechannel 1 with a detector, for example, a microscope as shown by FIG. 1,3, 5 or 6. By combining the microscope with a video camera or a CCDcamera, the progress of the migration of the cells can be automaticallyrecorded.

[0101] Although the cells passing through the channel 1 can be detectedand counted by directly observing the cells under the microscope, thedetection and counting can be easily performed by preliminarily labelingthe cells with a luminous or fluorescent substance and then capturingthe luminescence or fluorescence in a conventional manner.

[0102] As will be described hereinafter, the present invention makes itpossible to downsize the whole apparatus and thus samples can be treatedin microquantities. Moreover, it is possible to integrate multiplicityof units and thus a large number of specimens can be treated at the sametime. In addition, the treatment can be easily automated throughprogrammed control of suction and injection of liquids.

[0103] The whole apparatus involving the units for supplying andcollecting of cells, specimens and so on can be automated by combining aunit system made up of a single unit, an integrated unit consisting of aplural number of units of the same or different types or a plural numberof integrated units with a cell reservoir and a specimen reservoirtogether with, if needed, a pipette washing unit and sample supplypipette(s) for supplying cells or specimens which are mobile over theseunits, and further employing a mechanism for controlling the operationsof these pipettes. It is furthermore possible to control the detector sothat channels in a plural number of units are scanned along with thedetector repeatedly at definite intervals of time so as to detect thestates of the cells and trace the cell movements with the passage oftime. These controlling operations can be easily performed bycomputerized programming.

[0104] Next, the structure of the well unit according to the presentinvention will be illustrated in greater detail.

[0105] 1) Structure of Well Unit

[0106] As shown by FIGS. 2, 4 and 6, it is preferable that a channel andwells are integrally formed on a substrate 7. If needed, the substrate 7has holes (penetrating holes) for mounting tubes connected to respectivewells. The channel is provided with a bank 10. The upper part of thebank may be flat or, alternatively, provided with barriers 6constituting grooves 5 and, if needed, terraces 11.

[0107] By forming the bank 10 or forming the barriers 6 constituting thegrooves 5 on the bank 10, it is possible to easily establish the satewherein cells supplied into the wells are brought together in thevicinity of the channel and aligned in the flow direction of the cellsbefore the initiation of the migration. That is to say, by supplying acell suspension in one of the wells and then sucking an appropriateamount of the liquid from the well located in the opposite side acrossthe channel, cells are brought together in the vicinity of the channel.Owing to the bank 10 or the bank 10 and the barriers 6, the cells arealigned in the direction orthogonal to the flow direction. Subsequently,a chemotactic factor is injected into the well in the opposite side andthus the passage of the cells through the channel is initiated. In casewhere the cells are not brought together in the vicinity of channel ornot in the aligned state, the cells move irregularly and thus thechemotaxis can be hardly detected definitely due to the so-called randommovements of the cells. By using the well unit, an apparatus fordetecting chemotaxis of cells and separating cells may be fabricatedby: 1) placing a glass substrate 7 on a substrate 7 having wells and achannel (see FIG. 1); or 2) fastening a block 9 having tubes accessingto penetrating holes to the substrate 7 having wells and a channel insuch a manner that the tubes respectively accessing to the penetratingholes and then further pressing and fixing the glass substrate thereon(see FIGS. 3, 5 and 6). The block 9, the substrate 7 and the glasssubstrate 8 may be pressed and fixed by fastening with an O-ring (seeFIG. 39).

[0108] 2) Well

[0109] Wells 2 have a structure in which a cell suspension, a specimensolution such as a chemotactic factor-containing solution or aninhibitor-containing solution can be held in a resting state. Thisstructure is required for accurately detecting the movements of cells bytheir own actions. The capacity of the wells is not particularlyrestricted, so long as a liquid can be held therein in the minimumamount needed. To hold 0.3 μl a liquid, for example, wells of 0.1 mm indepth, 1.2 mm in width and 2.5 mm in length are usable.

[0110] 3) Connection Manner of Wells via Channel

[0111] To connect wells via a channel, use is commonly made of thedouble system as shown by FIGS. 2, 4, 5(2) and 6(2) or the triple systemas shown by FIGS. 7 and 8. FIGS. 2 and 7 show examples wherein nopenetrating hole is formed, while FIGS. 4, 6 and 8 show examples whereinpenetrating holes are formed. In the triple system, a relation amongthree substance can be examined at once by, for example, supplying acell suspension, a solution containing an inhibitor and another solutioncontaining a chemotactic factor respectively into the wells 2A, 2B and2C.

[0112] If needed, wells can be further jointed and connected. As theconnecting manner, use may be made of a so-called concentric type inwhich a plural number of wells are connected to each other around asingle well each via a channel as shown by FIGS. 9 to 11. Also, use canbe made of a concentric circular system as shown by FIGS. 12 and 13.FIGS. 12 and 13 show examples of concentric circular type of the triplesystem.

[0113] In the examples of FIGS. 9 to 11, a cell suspension is suppliedinto the central well 2A and various specimens are supplied into thewells 2B¹⁻⁴. Thus, a plural number of chemotactic factors can bedetected at the same time. By supplying a sample containing cells ofplural types into the well 2A, furthermore, the cells can be separateddepending on the types at once (i.e., sorting). For example, chemotacticfactors corresponding to respective cell types are put into the wells2B¹⁻⁴ and a sample containing plural types of cells (for example, wholeblood) is supplied into the central well 2A. Then the cells migratetoward the wells 2B¹⁻⁴ containing the corresponding chemotactic factors.After a definite time, the cells are collected from each of the wells2B¹⁻⁴.

[0114]FIG. 14 shows an example wherein a plural number of wells areconnected to each other each via a channel around a single well (2A)located at the center and, among these wells, at least a couple of wells(2B₁ and 2B₂) are connected to another common well (2C) each via achannel. In this case, a cell suspension is supplied into the well 2A, aspecimen solution containing a chemotactic factor is supplied into thewell 2C and specimen solutions containing different inhibitors aresupplied into the wells 2B₁ and 2B₂ respectively. Thus, the propertiesof the inhibitors can be compared and examined under the sameconditions.

[0115] 4) Specific Mode of Well Structure

[0116] When one of wells connected to each other via a channel (forexample, the well in which cells are to be held) or both of these wellsare provided with a wall orthogonal thereto so as to restrict the amountof a liquid or a cell suspension in the vicinity of the channel, thepositions of cells concerning the channel can be easily adjusted or theflow of a specimen sample can be easily controlled (FIG. 15). FIG. 15shows an example wherein wells 2A and 2B are connected to each other viaa channel 1 and walls 14A and 14B are formed in respective wellsorthogonally to the channel 1. When cells are injected into the well 2Avia a sample supplying tube 3A, a definite amount of cells are broughttogether between the wall 14A and the channel 1. Although the distancebetween the wall 14 and the channel 1 may be arbitrarily determined, itusually ranges from 50 to 300 μm.

[0117]FIG. 16 shows modification examples of the well unit having wallsprovided orthogonally to a channel. That is, FIG. 16(1) shows an examplewherein a channel is formed in a part of the well width; (2) shows anexample wherein a channel is halved at the center, a couple of wells(2B, 2C) are provided opposite to a single well (2A) across the channel,and a wall 14A is formed exclusively in the well 2A side; and (3) showsan example wherein two arrays of barriers are formed in both sides of aterrace 11 in a channel. Needless to say, these modifications are citedmerely by way of example and thus the present invention is notrestricted thereto.

[0118] 5) Channel

[0119] Now, examples of a channel 1 (FIGS. 3, 5, 6 and 8 to 16) will beillustrated by reference to FIGS. 1 to 6, 17 and 18. A channel 1 forms aspace, through which cells pass, between a bank 10 (a convex on asubstrate 7) partitioning wells 2A and 2B in both ends and a glasssubstrate 8. The upper part of the bank is flat (see FIG. 6) or providedwith barriers 6 constituting grooves 5 and, if needed, a terrace 11.FIG. 17 shows an example of the structure as shown by FIG. 1 in anapparatus, while FIG. 18 shows an example of the structure as shown byFIG. 3 or 5 in an apparatus.

[0120] The bank 10, which partitions the wells 2A and 2B located in bothends of the channel 1, is not particularly restricted in size. Forexample, the height of the bank 10 may range from about 0.03 to about0.1 mm, while the length in the direction toward the opposite well mayrange from about 0.01 to about 0.5 mm and the length in the directionorthogonal to the direction toward the opposite well may be the same asthe well width or shorter.

[0121] In case where no barrier constituting grooves is formed in theupper part of the bank, a gap or a depth fit for the diameter ordeformability of cells is provided between the upper face of the bankand the glass substrate. In this case, the depth usually ranges from 3to 50 μm depending on the type of cells. That is to say, the width mayrange from 3 to 10 μm (for example, 6, 7, 8 or 10 μm) in case ofneutrophils, eosinophils, basophils, monocytes/macrophages, T cells, Bcells and the like, and from 10 to 20 μm in case of cancer cells andcells existing in tissues.

[0122] 6) Barrier and Groove Constituted by Barrier in Channel

[0123] As FIG. 19 shows, barriers 6 may be formed on the upper face ofthe bank 10. Grooves 5 constituted by the barriers 6 may have anarbitrary cross-sectional shape, for example, a V-shaped section, aconvex section or a semicircular section (see FIG. 20).

[0124] The width of a groove 5 usually may range from 3 to 50 μm. It ispreferable that the width allows the passage of cells one by one. Thusan appropriate width may be selected depending on the cell type. Thewidth may range from 3 to 10 μm (for example, 6, 7, 8 or 10 μm) in caseof neutrophils, eosinophils, basophils, monocytes/macrophages, T cells,B cells and the like, and from 10 to 20 μm in case of cancer cells andcells existing in tissues.

[0125] The depth of the grooves 5 (i.e., the height of the barriers 6)may be appropriately determined depending on the depth of focus of amicroscope. Alternatively, the depth may be determined so as to allowthe passage of cells one by one. It is also possible to adjust both ofthe width and height of the grooves respectively to such levels asallowing the passage of cells one by one.

[0126] In case of adjusting the depth of the grooves 5 within the depthof focus of a microscope employed in observing the cell migration, adepth of about 4.5 μm is preferable at, for example, a focus depth of 10to 40× magnification, though the present invention is not restrictedthereto.

[0127] The number of the grooves 5 is determined depending on the widthof the barriers concerning the channel width and the groove width. Incase where the channel width is 1 mm, the barrier width is 10 μm and thegroove width is 5 μm, for example, the number of grooves is 66 at thelargest. To smoothly perform the detection and observation, the numberof the grooves preferably ranges from 1 to about 100, preferably fromabout 10 to about 70.

[0128] The length of the barriers 6 ranges from about 5 to about 400 μm.For example, use may be made of a barrier length of 5, 10, 20, 30, 40,60, 100, 200, 300 or 400 μm. The width of the barriers 6 per se can beappropriately determined. In case of employing the structure as will beshown by FIG. 25 hereinafter, it is effective that the width and lengthof the barriers are almost the same.

[0129] As FIGS. 21 and 22 show, the grooves 5 constituting the channel 1may be connected to each other via one or more grooves 12 orthogonal tothe direction toward the opposite well. Owing to this structure, cellsunder passage can be more accurately understood. In this case, the widthof the grooves 5 may be changed stepwise each time the grooves intersectgrooves 12 orthogonal thereto in the direction toward the opposite well,as shown by FIGS. 23 and 24. FIG. 23 shows an example wherein the widthof the barriers per se is changed. As FIG. 24 shows, it is also possiblethat the width of the grooves 5 is changed by increasing or decreasingthe number of the barriers 6 in the same size.

[0130] As FIG. 25 shows, grooves 5 in the direction toward the oppositewell may be formed by mutually shifting the positions thereof each timethe grooves intersect grooves 12 orthogonal thereto. FIG. 25 shows acase wherein the grooves 5 toward the opposite well are formed asshifting by ½ pitch each time the grooves intersect grooves 12orthogonal thereto, as in 5 a and 5 b. By forming the grooves 5 in thismanner, a specimen solution containing a chemotactic factor or aninhibitor can be sufficiently diffused. As a result, the specimensolution can be uniformly distributed in the direction toward theopposite channel and, at the same time, an increase/a decrease inpressure caused by the injection and collection of cells or specimenscan be efficiently avoided.

[0131] As FIG. 26 shows, the barrier may be jointed together in thedirection toward the opposite well.

[0132] 7) Positioning Mark in Channel

[0133] To detect the state of cells passing through a channel, adetector 13 returns onto a definite channel at definite intervals andthus the detection is repeated in some cases. For example, in anapparatus having an integration of a plural number of well units as willbe described hereinafter, the channels of respective well units arescanned along with the detector 13 in order to detect the state of cellspassing through the channels of respective units with the passage oftime. In such a case, it is convenient to give a screen-positioning markin a definite channel so that the same scope can be monitored on thescreen each time. The mark may be in any shape, so long as thepositioning can be facilitated thereby. Also, the mark may be given inany part, for example, in the upper part of the bank 10, in any part inthe terrace 11 as will be described hereinafter, or in the upper part ofone of the barriers. Either one or more marks may be provided (see FIGS.27 and 28(1)).

[0134] 8) Terrace in Channel

[0135] By providing a plane 11 on the upper face of the bank as shown byFIGS. 1 and 2, the passage of cells can be easily observed. (This planewill be referred to as a terrace.) It is preferable to provide thisterrace 11, though being not essentially required. In case of formingterraces 11 in both sides of arrays of the barriers 6 as shown by FIG.2, the length of the terraces in the direction toward the opposite wellmay appropriately range from about 0.03 mm to about 0.4 mm. As FIG. 27shows, one (11 ⁻¹) of the terraces (11 ⁻¹ and 11 ⁻²) formed in bothsides of the barrier arrays 6 may be longer than the other terrace (11⁻²). This structure makes it possible to easily observe cells havingpassed through the channel.

[0136] Although FIG. 27 shows an example wherein a mark (+) (23) isgiven on the upper face of the bank, this mark may be optionallyprovided.

[0137] It is also possible that a terrace is formed at the center of thebank and two arrays of barriers are provided in both sides of theterrace (see FIG. 28). By using this structure, cells having passedthrough the channel can be held on the terrace for a longer time, whichfacilitates the observation and counting of the cells. It is desirablethat the terrace located at the center has an area which can be includedin the microscopic field. FIG. 28(1) is a top plan view while FIG. 28(2)is a sectional view.

[0138] Although FIG. 28 shows an example wherein marks (23) are given attwo positions for facilitating positioning on screen, these marks may beoptionally provided.

[0139] FIGS. 29 to 31 show examples wherein terraces are formed in achannel in wells of the types shown by FIGS. 15 and 16. In each of FIGS.29 to 31, (1) is a top plan view while (2) is a sectional view of thepart indicated by a broken line in (1) FIG. 29 shows an example whereinterraces 11A and 11B are formed in both sides of a channel to walls 14Aand 14B provided orthogonally to the channel. FIG. 30 shows an examplewherein a terrace 11A is formed to a wall 14A orthogonal to a channelexclusively in one side of the channel, while a terrace not extended toa wall 14B is formed in the other side. FIG. 31 shows an examplewherein, in a case of forming a wall 14A orthogonally to a channelexclusively in the side of a well 2A into which cells are injected, aterrace 11A is provided to the wall 14A. By forming such a terrace in awell unit of the type as shown by FIG. 15 or 16, the rapid diffusion ofa chemotactic factor or an inhibitor can be prevented after the passagethereof from the well 2B to the well 2A via the channel. In case offorming no such a terrace, the diffusion proceeds rapidly due to thelarge volume in the vicinity of the channel.

[0140] By forming a multistage bank 10 (i.e., forming multistageterraces 11 of the bank 10) as shown by FIG. 32, cells put into a wellin one side can be easily brought together in the vicinity of the bank10 by sucking from the other side. In case where the cells areneutrophils, eosinophils, basophils, etc., for example, the distancebetween the terraces 11 ⁻² and 11 ⁻³ and a glass substrate 8 (i.e.,corresponding to the height of a barrier 6 in the figure) is set to 3 μmand the distance between the terraces 11 ⁻¹ and 11 ⁻⁴ and a glasssubstrate 8 is set to 4.5 μm. Then cells are supplied into a well 2A andthe liquid is sucked from the side of another well 2B. In this case, thecells once stop at the terrace 11 ⁻¹. Next, the cells are liable to bebrought together between the terrace 11 ⁻² and the glass substrate 8.The distance between each of the terraces 11 ⁻¹ to 11 ₄ and the glasssubstrate 8 can be arbitrarily determined depending on the cells to betreated. Although these distances usually range from 3 to 5 μm, thepresent invention is not restricted thereto. When the terrace (11 ⁻³) inthe side opposite to the well containing the cells is made about 1.5 to5 times longer than the terrace (11 ⁻²) in the side of the wellcontaining the cells, the cells having passed through the channel can bemore easily observed and counted.

[0141] 9) Obstacle in Channel

[0142] As an example of the structure wherein, before supplying achemotactic factor, cells are aligned forward along the start line in awell in the other side under the same conditions, it is proposed to formobstacles for controlling the migration of cells in a channel.

[0143] The “obstacles” as used herein do not completely block butrestrict the cell migration. Although an array of convexes and an arrayof triangular prisms or quadratic prisms may be cited as examples of theobstacles, they may be in any shape so long as the above object can beachieved thereby. It is favorable that the obstacles are formed in theupper part of the bank, though the present invention is not restrictedthereto so long as the object can be achieved. In case where the wholeupper face of the bank serves as a terrace without any barrier, theobstacles may be formed close to an end thereof (see FIG. 40(1)). Incase where barriers and a terrace are formed on the upper face of thebank, the obstacles may be formed in the well side of the terrace inparallel to the barrier array(s) (see FIG. 40 (2) to (4)).

[0144] In FIG. 40 (1), (2) and (4), an array of convexes is employed asthe obstacles. In FIG. 40(3), an array of triangular prisms is employedas the obstacles.

[0145] The height of the obstacles may be the same as the length fittingfor the diameter or deformability of cells or amounts to ¼ to ½ thereof.The intervals among the obstacles may be the same as the length fittingfor the diameter or deformability of cells. In case where the obstaclesare lower, the intervals may be shortened.

[0146] 10) Arrangement of Multiplicity of Units

[0147] By referring a plural number of wells connected to each othereach via a channel as a single unit, a plural number of units may bearranged and integrated. Thus, a well unit whereby a large number ofspecimens can be treated at the same time can be obtained. Thearrangement and integration can be made in various types depending onthe purpose, for example, units of the same type are arranged inparallel (e.g., FIGS. 33 to 35), or circularly (e.g., FIG. 36), or unitsof different types are arranged (e.g., FIG. 37). Next, the types of thearrangement and integration will be described by reference to respectivefigures. However, it is to be understood that the present invention isnot construed as being restricted thereto and thus various combinationsmay be also employed depending on the purpose.

[0148]FIGS. 33 and 34 show examples wherein 12 well units each having acouple of wells connected via a channel as shown by FIG. 3 are mountedon a square substrate 7 (16 mm×16 mm). The units are each 5.7 mm in themajor sides and 1.2 mm in the minor sides and located at intervals of0.8 mm. In the example of FIG. 33, square penetrating holes 3 a and 3 bare formed in the substrate 7, while round penetrating holes are formedin the example of FIG. 34.

[0149]FIG. 35 shows an, example wherein 12 well units of the type asshown by FIG. 15 are mounted on a substrate 7.

[0150]FIG. 36 shows an example wherein independent double system wellunits are integrated circularly. Although each well has penetratingholes in the example of FIG. 36, it is needless to say that some wellsmay have no penetrating hole. Concerning the size, for example, thewidth of wells 2A and 2B in the radial direction is 1.5 mm, the channelwidth is 0.5 mm and the groove width is 10 μm. In this case, the radiusof the whole unit is 5.0 mm. As a matter of course, the size can bechanged depending on the purpose.

[0151]FIG. 37 shows an example wherein the integrations composed ofmultiplicity of units as shown by FIGS. 33 to 36 are further integrated.In FIG. 37, squares represented by A¹⁻⁴, B¹⁻⁴, C¹⁻⁴ and D¹⁻⁴respectively correspond to the integrations of well units of FIGS. 33 to36. In this case, the arrays A, B, C and D are integrations of units ofdifferent types.

[0152] In case of integrating multiplicity of units, a single block 9can be provided so as to connect tubes to all of the units. Similarly, asingle glass substrate 8 can be used as a whole.

[0153] 11) Construction of Well and Channel

[0154] As a material of the substrate 7, it is preferable to usesingle-crystal silicon which can be easily fine processed and isrelatively inert to cells. The barriers 6 and the grooves in the channel1 can be constructed by subjecting the single-crystal silicon tophotolithography or etching (for example, wet etching or dry etching)employed in manufacturing integrated circuits. The wells 2 and thepenetrating holes 3 a and 4 a, which are larger than the barriers 6 andthe grooves 5, can be constructed by using various known engineeringtechniques such as sand blasting and dry etching. In addition tosingle-crystal silicon, use can be made of hard glasses, hard plastics,metals, etc., so long as a microstructure can be constructed in thechannel. In case of using plastics, it is preferable to employ atreatment for making the surface hydrophilic, for example, forming ahydrophilic film on the surface. It is also possible to separatelyconstruct the channel 1 and the wells 2 and then combine them together.

[0155] Now, an example of the production process by wet etching will beillustrated by reference to FIG. 38. First, grooves 5 are formed in apart of a single-crystal silicon substrate (1) as shown in (2) and (3),wherein (2) is a top plan view while (3) is a sectional view along thebroken line. Next, the whole construct excluding the grooves 5 and thebarriers 6 is cut downward by the height of the barrier (for example,4.5 μm) as shown in (4). Subsequently, the construct is further cutdownward, while a bank 10 is left at the center to form wells 2A and 2Bas shown in (5) If necessary, penetrating holes 3 a and 4 a are formedat the bottom of the wells by sand blasting or the like, as shown in(6). (7) is a top plan view of the construct of (6). A substrate havingintegrated well units can be constructed in the same manner.

[0156] 12) Fabrication of Apparatus for Detecting Chemotaxis of Cellsand Separating Cells

[0157] An apparatus for detecting chemotaxis of cells and separatingcells with the use of the well unit according to the present inventioncan be fabricated as follows. An apparatus of the type as shown by FIG.1 can be fabricated by combining a substrate 7 with a glass substrate 8,while an apparatus of the type as shown by FIGS. 3 and 5 can befabricated by combining a substrate 7, a glass substrate 8 and a block9.

[0158] As shown by FIGS. 3, 5 and 6, the block 9 is a member havingtubes connected to wells. If mechanically possible, the tubes can bedirectly mounted to the penetrating holes 3 a and 4 a of the wells. Inthis case, no block is needed. The tubes 3 and 4 usually have a squareor round cross-sectional shape. Although these tubes are not restrictedin size, a square tube has a side length of about 1 mm while a roundtube has a diameter of about 1 mm in usual. To hold a cell suspension ora specimen solution in a desired volume, it is necessary that thesetubes have a length of about 2 to 10 mm.

[0159] The materials of the block or tubes may be selected from amongglasses, plastics such as acrylic resins and metals. The block and tubescan be easily produced by using commonly employed engineering techniquessuch as mechanical drilling or laser drilling. Alternatively, the blockand tubes can be produced by irradiating a photopolymer resin with lightand then eliminating the unsensitized parts by dissolving in a solventwhile leaving the sensitized parts.

[0160] As shown by FIGS. 1, 3, 5 and 6, the glass substrate 8 is tightlypressed on the substrate 7 to provide a space in which a liquid iscontained, thereby enabling the observation of cells passing through thechannel. Thus, the glass substrate 8 should remain optically transparentand flat. It is also favorable that cells adhere to the glass substrate8. Use can be made therefor of glass and plastics such as transparentacrylic resins, so long as the above objects can be achieved thereby.Its thickness adequately ranges from 1 to 2 mm, though the presentinvention is not restricted thereto.

[0161]FIG. 39 shows an example of the fabrication of an apparatus fordetecting chemotaxis of cells and separating chemotactic cells by usingthe well units according to the present invention. A substrate havingwell units formed thereon, a packing 16 and a block 9 covering it areplaced between a cover cap 17 and an intermediate base 21. A glasssubstrate 8 is placed between the intermediate base 21 and a bottom base22 and fastened with screws. The locations of the block 9 and thesubstrate 7 are specified by the intermediate base 21 and fixed by guidepin receiver holes 19 provided at the bottom face of the block.Alternatively, the substrate 7 may be directly pressed and fixed to theblock 9.

[0162] 13) Detection Means

[0163] The detection means to be used in the present invention is ameans of detecting cells which are passing through a channel or havepassed therethrough. If necessary, it involves a means of recording thedetection data. Any means known as a means of detecting and recordingcells is usable therefor. Use can be made of, for example, a microscopeoptionally combined with a video camera. It is also possible to employ asystem having an objective lens provided with a CCD camera. For thedetection in integrated units, it is preferable to employ a systemwherein the channels of the units are successively scanned along with anobjective lens.

[0164] As shown by FIGS. 1, 3, 5 and 6, the detection means is providedin a channel of a unit. In an apparatus having multiplicity of unitsintegrated together, it is also possible to employ a system wherein thedetector moves successively over the arrays of the units for detectionand recording. In this case, the channels of the aligned units arescanned with the detector. Thus, the detection in each channel can becarried out at definite intervals of time and the movements of cells canbe monitored with the passage of time. Either one or more scanningdetectors may be employed. Owing to this constitution, a relativelysmall number of detectors suffice for the detection in multiplicity ofintegrated units.

[0165] Cells which are passing or have passed through a channel can bedetected and counted by directly observing the cells with a microscope,a CCD camera, a CCD video camera, etc. Alternatively, the detection andcounting can be easily performed by preliminarily labeling the cellswith a luminous or fluorescent substance and then capturing theluminescence or fluorescence in a conventional manner.

INDUSTRIAL APPLICABILITY

[0166] Use of the well unit according to the present invention makes itpossible to fabricate an apparatus for detecting chemotaxis of cells andseparating cells appropriate for various purposes. For example, anapparatus which scarcely suffers from a pressure change (an increase inpressure) in the horizontal direction at the step of injecting a sampleand thus shows little migration of specimens or cells due to externalpressure can be obtained thereby. By using such an apparatus wherebymovements of cells by their own actions can be accurately understood,quantitative and qualitative data certainly reflecting the effect of achemotactic factor or an inhibitor and the properties of cells can beobtained.

[0167] In a Boyden chamber, random movements of cells are also capturedand thus the background without any chemotactic factor becomes high. Inan apparatus with the use of the well unit according to the presentinvention, in contrast thereto, a background of almost zero can beestablished and thus a high quantitative accuracy can be achieved.

[0168] The well unit according to the present invention is suitable fortreating samples in microquantities. Namely, samples can be used in anamount {fraction (1/10)} to {fraction (1/1000)} times as much in theconventional cases with the use of a Boyden chamber. By using wholeblood as a sample, for example, measurement can be made by using 0.1 ↑lof blood in case of detecting the chemotaxis of neutrophils and about 1μl of blood in case eosinophils, monocytes or basophils.

[0169] Moreover, the well unit according to the present invention can bein a microsize and thus multiplicity of the units can be integratedtogether, which brings about a merit that an apparatus whereby a largenumber of samples can be simultaneously treated can be fabricated.

[0170] The well unit according to the present invention is suitable formoving definite cells from a cell suspension containing plural types ofcells and then collecting the definite cells from a well. Thus, targetcells can be surely collected.

[0171] In the well unit according to the present invention, movements ofindividual cells can be easily understood by forming a bank in a channel1, by providing the bank with grooves 5 in various modes having a widthand/or a depth fit for the diameter or deformability of cells, or byproviding the bank with a plane so as to give a gap fit for the diameteror deformability of cells. By forming the bank or by providing the bankwith barriers constituting grooves, moreover, it is possible to easilybring together the cells held in the well in the vicinity of the channeland align them in the flow direction of the cells before the initiationof migration, which enhances the accuracy in detecting the chemotaxis ofthe cells.

[0172] In the well unit according to the present invention, thechemotaxis of a part of blood cells among various cells in a samplecontaining plural types of cells (for example, whole blood) can beexamined without preliminarily separating them. By selecting appropriatechemotactic factors, furthermore, cells in a sample containing pluraltypes of cells can be classified depending on the types.

1. A well unit to be used in an apparatus for detecting chemotaxis ofcells and separating cells characterized in that a plural number ofwells, in which a liquid sample can be held in a resting state, areconnected to each other via a channel, the channel is provided with abank, the wells are formed so as to tightly bond to a glass substrate,and, in the upper part of the bank, barriers constituting one or moregrooves having a width and/or a depth fit for the diameter ordeformability of cells are provided, or a plane is provided so as togive a gap fit for the diameter or deformability of cells between theplane and the glass substrate.
 2. The well unit as claimed in claim 1characterized in that a plural number of wells, in which a liquid samplecan be held in a resting state, are connected to each other via achannel, the channel is provided with a bank and, in the upper part ofthe bank, barriers constituting one or more grooves having a widthand/or a depth fit for the diameter or deformability of cells areprovided.
 3. The well unit as claimed in claim 1 characterized in that aplural number of wells are connected in series to each other via achannel.
 4. The well unit as claimed in claim 1 characterized in that aplural number of wells are connected to a single well each via achannel.
 5. The well unit as claimed in claim 4 characterized in that,among a plural number of wells connected to a single well each via achannel, at least two wells are connected to another common well eachvia a channel.
 6. The well unit as claimed in claim 1 characterized inthat a plural number of wells are wells holding a cell suspension andwells holding a solution containing a chemotactic factor.
 7. The wellunit as claimed in claim 1 characterized in that a plural number ofwells are wells holding a cell suspension, wells holding a solutioncontaining a chemotactic factor, and wells holding a solution containinga chemotactic factor inhibitor.
 8. The well unit as claimed in claim 1characterized in that a wall orthogonal to the channel is provided inone or both of wells connected to each other via the channel to restrictthe amount of the liquid in the vicinity of the channel.
 9. The wellunit as claimed in claim 8 characterized in that a terrace is formed toone or both of the walls formed orthogonally to the channel.
 10. Thewell unit as claimed in claim 1 characterized in that ascreen-positioning mark for detecting cells is given on any point in theupper part of the bank.
 11. The well unit as claimed in claim 1characterized in that a multistage bank is formed in the channel. 12.The well unit as claimed in claim 2 characterized in that the groovesformed in the channel are connected to each other via one or moregrooves orthogonal to the direction toward the opposite well.
 13. Thewell unit as claimed in claim 12 characterized in that the width of aplural number of grooves in the direction toward the opposite well inthe channel is changed stepwise each time the grooves intersect one ormore grooves orthogonal thereto.
 14. The well unit as claimed in claim12 characterized in that a plural number of grooves in the directiontoward the opposite well in the channel are formed by mutually shiftingthe positions thereof each time the grooves intersect one or moregrooves orthogonal thereto.
 15. The well unit as claimed in claim 2characterized in that terraces are formed in the front and the rear ofan array of barriers constituting one or more grooves having a widthand/or a depth fit for the diameter or deformability of cells in thechannel and the terrace in the cell flow direction is longer than theother terrace.
 16. The well unit as claimed in claim 2 characterized inthat a terrace is formed at the center in the channel, arrays ofbarriers constituting one or more grooves having a width and/or a depthfit for the diameter or deformability of cells are formed at twopositions in both sides of the terrace, and, if desired, terraces arefurther formed outside the barrier arrays.
 17. A well unit as claimed inclaim 1 characterized in that obstacles for controlling the migration ofcells in the step of aligning the cells along the start line are formedorthogonally to the direction toward the opposite well.
 18. A well unitas claimed in claim 2 characterized in that obstacles for controllingthe migration of cells in the step of aligning the cells along the startline are formed in parallel to the arrays of barriers.
 19. A well unitto be used in an apparatus for detecting chemotaxis of cells andseparating cells characterized in that the well units as claimed inclaims 1 to 18 are each referred to as a single unit and a plural numberof units of one or more types are integrated.